Involvement of P21 WAF1/CIP1 and P27 KIP1 in troglitazone-induced cell cycle arrest in human hepatoma cell lines

Abstract

Peroxisome proliferator-activated receptor-γ (PPAR-γ) regulates cell growth and differentiation. Recent evidence has suggested that PPAR-γ ligands have antitumor effects through inhibiting cell growth and inducing cell differentiation in several types of malignant neoplasm. In the present study, we investigated (a) the expression of PPAR-γ in both human hepatoma cell lines and five resected human hepatocellular carcinoma (HCC) tissues, (b) the growth inhibitory effect of troglitazone, a PPAR-γ ligand, on those hepatoma cells, and (c) the molecular mechanisms of troglitazone-induced cell cycle arrest. Five hepatoma cell lines, HLF, HuH-7, HAK-1A, HAK-1B, and HAK-5, were used. The mRNA expression levels of PPAR-γ, p21 WAF1/CIP1 and p27KIP1 were determined by real-time quantitative reverse transcription-polymerase chain reaction. The expression of cell cycle-regulating proteins, such as p21, p27, cyclin E, and pRb, was examined using Western blotting. PPAR-γ was constitutively expressed in all the cell lines and the HCC tissues used in this study. A cytostatic effect of troglitazone was found in those cell lines, and this inhibition of cell growth was dosage dependent. G0/G1 arrest was demonstrated in flow cytometric analysis in HLF, HAK-1A, HAK-1B, and HAK-5, all of which showed an increased expression of p21 protein. However, HuH-7, lacking p21 protein expression, did not demonstrate clear arrest in the cell cycle analysis. HLF, which is pRb deficient, responded most profoundly to troglitazone, showing an increased expression of not only p21 but also p27. These findings suggest that p21 and p27 might be involved in troglitazone-induced cell cycle arrest in human hepatoma cells.